Discovery of the order 'Quisvirales' redefines the evolution of RNA replication and transcription in the phylum Pisuviricota.

Abstract

Genome replication in positive-stranded RNA (ssRNA+) viruses is mediated by cognate enzymes, including ubiquitous RNA-dependent RNA polymerase (RdRp). In ssRNA+ viruses with multiple open reading frames (ORFs) in their genomes, replication often is accompanied by synthesis of subgenomic RNAs (transcription) for expression of 3'-proximal ORFs. In addition, all ssRNA+ viruses with genomes larger than ~7 kb encode helicases, linking helicases to RNA genome expansion. Helicases are essential ATPases that unwind nucleic acids and are classified into six recognized superfamilies (SF1-SF6). In the phylum Pisuviricota that includes important pathogens, helicases of SF1-SF3 are integrated into multi-enzyme replicase polyprotein(s) including 3C(-like) protease (3CLpro) and RdRp. Here, large-scale mining of invertebrate metatranscriptomes and targeted genome sequence assembly uncovered six spider-associated ssRNA+ viruses that, based on their conserved 3CLpro-RdRp module in replicase polyproteins, genome size (20-22 kb), and phylogeny, form a family-like cluster in a putative order, named 'Quisvirales'. Quisviruses have similar genome and replicase architectures to enveloped coronaviruses and other nidoviruses. Notably, quisviruses encode ORFs 1a and 1b with predicted -1 programmed ribosomal frameshifting elements in the ORF1a/b overlap region. Using an original mapping approach for detecting chimeric sequencing reads, we obtained evidence that 3'-proximal ORFs are expressed via 5'-coterminal, leader-containing subgenomic RNAs. This suggests that the quisvirus subgenomic RNAs are generated through discontinuous transcription-a mechanism otherwise exclusively found in nidoviruses among the many ssRNA+ virus orders that synthesize subgenomic RNAs. Striking differences between nido- and quisviruses are, however, the RdRp being the only common core ORF1b-encoded enzyme and the replacement of the nidovirus SF1 helicase by a novel superfamily helicase. This quisvirus SF7 helicase, like the Picornavirales SF3 helicase, comprises an AAA+ (ATPase-like) domain typical for ring-forming helicases and thus must play an essential role in replication. The discovery of the order 'Quisvirales' demonstrates that viruses employing large replicase polyproteins of nidovirus-like complexity and discontinuous transcription may have evolved repeatedly from an 3CLpro-RdRp-encoding ancestor.